NER and HR Pathways Act Sequentially to Promote UV-C-Induced Germ Cell Apoptosis in Caenorhabditis Elegans

Home , RAD51, XPB

Cell Death and Differentiation (2011) 18, 897–906 & 2011 Macmillan Publishers Limited All rights reserved 1350-9047/11 www.nature.com/cdd NER and HR pathways act sequentially to promote UV-C-induced germ cell apoptosis in Caenorhabditis elegans

L Stergiou1,3,4, R Eberhard1,2,4, K Doukoumetzidis1 and MO Hengartner*,1

Ultraviolet (UV) radiation-induced DNA damage evokes a complex network of molecular responses, which culminate in DNA repair, cell cycle arrest and apoptosis. Here, we provide an in-depth characterization of the molecular pathway that mediates UV-C-induced apoptosis of meiotic germ cells in the nematode Caenorhabditis elegans. We show that UV-C-induced DNA lesions are not directly pro-apoptotic. Rather, they must ﬁrst be recognized and processed by the nucleotide excision repair (NER) pathway. Our data suggest that NER pathway activity transforms some of these lesions into other types of DNA damage, which in turn are recognized and acted upon by the homologous recombination (HR) pathway. HR pathway activity is in turn required for the recruitment of the C. elegans homolog of the yeast Rad9-Hus1-Rad1 (9-1-1) complex and activation of downstream checkpoint kinases. Blocking either the NER or HR pathway abrogates checkpoint pathway activation and UV-C-induced apoptosis. Our results show that, following UV-C, multiple DNA repair pathways can cooperate to signal to the apoptotic machinery to eliminate potentially hazardous cells. Cell Death and Differentiation (2011) 18, 897–906; doi:10.1038/cdd.2010.158; published online 10 December 2010

Eukaryotic cells possess several surveillance mechanisms the signaling network that ultimately balances cellular DNA that, upon sensing DNA damage, initiate signaling cascades damage responses between genome maintenance, senes- that lead to response programs such as cell cycle arrest, DNA cence and death. Accordingly, loss of their function has repair and apoptosis, to protect the organism against the consequences for DNA repair, cellular proliferation and introduction of new mutations. Disruption of such pathways survival.6–8 results in increased genomic instability, a hallmark of most Simple model organisms are very useful to decipher types of cancers.1,2 complex DNA damage responses. In Caenorhabditis elegans, Genetic and biochemical studies have provided a thorough the effects of IR have been studied extensively.9,10 We mechanistic understanding of the various repair processes previously reported a genetic pathway that induces both initiated upon recognition of specific types of lesions. For apoptotic cell death of meiotic cells and cell cycle arrest of example, the nucleotide excision repair (NER) pathway proliferating mitotic cells following UV-C treatment.11 We removes cyclobutane pyrimidine dimers (CPDs) and 6-4 identified several new genes required for these responses and photoproducts generated upon exposure to ultraviolet (UV-C) genetically ordered them into a signaling pathway that light, whereas the homologous recombination (HR) machin- overlaps with, but is distinct from the pathway(s) activated ery repairs double-strand DNA breaks (DSBs) induced by upon IR. treatments such as ionizing radiation (IR).3 In this study, we investigate the molecular mechanism by Mutations in NER components underlie the syndromes which UV-C triggers apoptosis in the C. elegans adult xeroderma pigmentosum (XP), trichothiodystrophy or Cock- hermaphrodite germ line. We show that lesions caused by ayne syndrome. Patients are hypersensitive to sunlight and UV-C are not pro-apoptotic per se; rather, they first require exhibit a variety of clinical features, including developmental processing by the NER machinery before they can activate defects, predisposition to skin cancer or internal tumors, apoptosis. A fraction of UV-C lesions is likely transformed by neurological disorders and highly accelerated aging.4 Studies NER into DNA intermediates that are substrates for the HR in cell culture and mouse models,5,6 as well as in yeast have machinery. Activation of the latter, in turn, leads to recruitment led to the detailed molecular characterization of the NER of the C. elegans homolog of the yeast Rad9-Hus1-Rad1 factors.3 Besides repair, many of these factors participate in (9-1-1) complex, activation of downstream checkpoint kinases

1Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, Zurich 8057, Switzerland and 2PhD Program in Molecular Life Sciences, Life Science Zurich Graduate School and MD/PhD Program, University of Zurich, Zurich, Switzerland *Corresponding author: MO Hengartner, Institute of Molecular Life Sciences, University of Zurich, Winterthurerstrasse 190, Zurich 8057, Switzerland. Tel: þ 41 44 635 3140; Fax: þ 41 44 635 6861; E-mail: [email protected] 3Current address: Pike Pharma, Schlieren-Zurich, Switzerland. 4These authors contributed equally to this work. Keywords: apoptosis; C. elegans; HR; NER; UV-C Abbreviations: UV-C, ultraviolet light C (254 nm); IR, ionizing radiation; CPDs, cyclobutane pyrimidine dimers; DSB, double-strand DNA break; NER, nucleotide excision repair; HR, homologous recombination; 9-1-1 complex, Rad9-Hus1-Rad1 complex (HPR-9/HUS-1/MRT-2 in C. elegans); XPA, XPB, XPD, XPF, XPG, xeroderma pigmentosum complementation group A, B, D, F, G; RPA-1, replication protein A large subunit homolog in C. elegans Received 06.5.10; revised 07.9.10; accepted 20.9.10; Edited by E Baehrecke; published online 10.12.10 HR is required for UV-C-induced apoptosis in C. elegans L Stergiou et al 898

and initiation of p53-dependent apoptosis. Our results indicate rad-54(RNAi);spo-11(ok79) animals showed a greatly that UV-C-induced apoptosis in C. elegans requires the reduced response to IR and, surprisingly, also to UV sequential activation of at least two distinct DNA damage (Figure 1g). Importantly, spo-11(ok79) mutants respond response pathways. normally to both IR and UV, showing that the DNA damage response pathways are functional (Figure 1g). The HR pathway therefore plays an important role in promoting Results apoptosis in response to UV-C. Finally, we tested animals lacking the recombinase Components of the HR pathway are required for RAD-51. In the human and yeast homologs, RAD-51 acts UV-C-induced apoptosis. We previously showed that in downstream of MRE-11 and RAD-54 to promote strand C. elegans UV-C radiation induces cell cycle arrest in mitotic exchange during HR. rad-51(RNAi) animals also fail to germ cells and apoptosis of meiotic cells at the pachytene complete meiotic recombination, resulting in increased basal stage.11 We also reported that XPA-1 and XPC-1, two levels of apoptosis (Figure 1h). However, unlike in rad-54 components of the NER pathway, are required for the mutants, this increase can be suppressed only by activation of these responses, with XPA-1 acting upstream of atl-1(tm853), but not atm-1(gk186) (Supplementary Figure 2). the 9-1-1 complex protein HUS-1 and the p53 homolog In contrast to mre-11 and rad-54 mutants, rad-51(lf) CEP-1 in UV-induced apoptosis.11 animals did not show any defect in either UV- or IR-induced How does XPA-1 function promote p53 activation following apoptosis (Figure 1h).15 Moreover, UV-C treatment led to UV-C exposure? One possibility is that NER processes a RAD-51 foci formation (Supplementary Figure 3a), indepen- fraction of UV-C-induced damage into other types of DNA dent of both xpa-1 and rad-54 (Supplementary Figures 3b and structures, which might in turn activate apoptosis. Given our c). These observations suggest that UV-C-induced RAD-51 previous observation that many genes involved in response to foci formation and apoptosis are independent events, and that DSBs are also activated following UV-C, we focused our initial RAD-51 is not essential for UV-C-induced apoptosis. attention on the HR pathway. mre-11(ok179) mutants, which Taken together, our results support the idea that the lack the exonuclease responsible for the initial processing of proteins involved in early stages of HR also act to promote DSBs during meiosis, showed a compromised apoptotic apoptosis in response to UV-C damage. response to UV-C, suggesting a requirement for MRE-11 in the initiation of UV-C-induced apoptosis (Figure 1a). By The HR pathway acts downstream of the NER pathway to contrast, IR-induced apoptosis was only slightly reduced promote UV-C-induced apoptosis (Figure 1a), in agreement with previous reports,12 possibly owing to alternative processing pathways. The data above suggest that both NER and HR pathways are The homologs of RAD-54 and RAD-51 functionally interact required for UV-C-induced apoptosis. These two pathways during recombinational repair in yeasts and mammals.13,14 might act in parallel and be simultaneously required for the We analyzed two previously uncharacterized, likely null induction of apoptosis. Alternatively, they might act in succes- rad-54 mutants, tm1268 and ok615 (Supplementary Figure sion, with NER recognizing and processing the initial UV-C 1a). As homozygous rad-54 progeny from heterozygous lesions and HR relaying the signal to the apoptotic machinery. To mothers show fully penetrant maternal-effect lethality, we distinguish between these two possibilities, we determined the analyzed ﬁrst-generation homozygotes for germline apopto- effect of mutations in the NER or HR pathways on the subcellular sis. Under normal growth conditions, both rad-54(ok615) and localization of selected DNA damage response proteins. rad-54(tm1268) mutants exhibit a strong increase in germline We started by examining the distribution of a functional apoptosis, an effect that could be phenocopied by RNAi RAD-54::YFP fusion protein (Supplementary Figure 1b). (Figure 1b). This increase was dependent on the endonu- RAD-54::YFP formed foci following exposure to both IR or clease SPO-11, which speciﬁcally generates the DSBs that UV-C, consistent with our observation that the HR pathway initiate meiotic recombination (Figure 1c),12 suggesting that participates in UV-C-induced responses (Figure 2a). In xpa-1 the increased germ cell death is due to the accumulation of and xpc-1 mutants, foci accumulation following UV treatment, unresolved recombination intermediates. rad-54(rf)-induced but not IR, was dramatically reduced (Figure 2b), suggesting apoptosis was abrogated in atl-1(tm853) or cep-1(RNAi) that the NER component XPA-1 acts upstream of RAD-54 animals, and reduced in atm-1(gk186) mutants (Figures 1d recruitment in the UV-C response pathway. and e). These results suggest that rad-54(lf) mutants We previously showed that xpa-1 or xpc-1 mutants exhibit accumulate DSBs, which activate apoptosis in an ATL-1/ increased basal levels of apoptosis in the germ line,11 ATM-1- and CEP-1-dependent manner. probably because accumulation of unrepaired endogenous Interestingly, exogenous damage by X-rays or UV failed damage leads to apoptosis through the activation of (an)other to further increase the levels of germ cell apoptosis in DNA damage signaling pathway(s). Interestingly, the basal rad-54(ok615) mutants (Figure 1f), suggesting either that levels of RAD-54::YFP foci were also elevated in the absence the DNA damage response pathway (or the apoptotic of xpa-1 (Figure 2b), suggesting that the HR pathway machinery to which it signals) is already ‘saturated’ by the mediates the increased apoptosis observed in xpa-1 mutants. strong signal generated by the unresolved meiotic intermedi- Consistent with this hypothesis, mutations in genes that act ates in rad-54 mutants or that the HR pathway is required downstream of DNA DSBs, such as hus-1 and atl-1 for IR- and UV-induced apoptosis. To address this issue, (Supplementary Table S1), as well as atm-1 and cep-111 we blocked SPO-11 endonuclease function. We found that abrogated the apoptosis phenotype of xpa-1 mutants.

Cell Death and Differentiation HR is required for UV-C-induced apoptosis in C. elegans L Stergiou et al 899

Figure 1 Several components of the HR repair pathway are required for UV-C-induced germ cell apoptosis. (a) mre-11 mutants fail to induce apoptosis in response to UV-C. Staged young adult mre-11(ok179) animals were treated with either 100 J/m2 UV-C or 120 Gy X-rays and germ cell corpses were scored at the indicated time points. (b–e) Genetic characterization of rad-54 mutants. (b) rad-54(lf) results in increased levels of germ cell apoptosis. Germ cell corpses were scored every 12 h until 36 post the L4/adult molt in staged rad-54(ok615) or rad-54(tm1268) mutants, or rad-54(RNAi)-treated animals. (c–e) rad-54(lf)-induced germ cell death depends on spo-11 (c), cep-1 (d) and ATM/ATR function (e). (f) rad-54 mutants fail to induce apoptosis in response to UV-C and IR. Staged young adult rad-54(ok615) animals were treated with either 100 J/m2 UV-C or 120 Gy X-rays and germ cell corpses were scored at the indicated time points. (g) Loss of spo-11 function does not restore UV-C- and IR-induced apoptosis in rad-54(RNAi) animals. Staged spo-11(ok79) L1 larvae were raised on bacteria expressing rad-54 or gfp dsRNA, and were treated with either 100 J/m2 UV-C or 120 Gy X-rays as young adults. Germ cell corpses were scored at the indicated time points. (h) rad-51(lf) animals normally induce apoptosis in response to UV-C. Staged wild-type L1 larvae were raised on bacteria expressing rad-51 or control dsRNA, and were treated with either 100 J/m2 UV-C or 120 Gy X-rays as young adults. Germ cell corpses were scored at the indicated time points. Data shown in all cases represent the average±S.D. of two or three independent experiments (n420 animals for each experiment)

We also examined the localization of RAD-54::YFP in mechanisms for RAD-54 recruitment to sites of damage might atm-1, hus-1 and mre-11 mutants. Foci formation was exist, which differ in their requirement for MRE-11. Interest- essentially wild type in atm-1 mutants and only mildly affected ingly, Hayashi and co-workers16 have shown a similar in hus-1 mutants, indicating that ATM-1 and HUS-1 are either difference in the requirement of RAD-50 for RAD-51 loading not required for or act downstream of RAD-54 recruitment to onto sites of DSBs. sites of DNA damage. By contrast, UV-C-treated mre-11 Next, we examined the distribution of the replication protein animals failed to show any focal accumulation of RAD- A subunit homolog in C. elegans (RPA-1). RPA is a 54::YFP in the mid–late pachytene cells (Figure 2b), whereas heterotrimeric single-stranded DNA-binding protein highly IR-treated animals exhibited wild-type foci numbers through- conserved in eukaryotes that plays an important role in DNA out the gonad (Figure 2b). Therefore, two (or more) distinct replication and repair.17–20 Transgenic animals expressing

Cell Death and Differentiation HR is required for UV-C-induced apoptosis in C. elegans L Stergiou et al 900

Figure 2 The HR pathway acts downstream of the NER pathway to induce UV-C-induced germ cell apoptosis. (a and c) Fluorescent microscopy of mid–late pachytene germ cells expressing RAD-54::YFP (a) or RPA-1::YFP (c). Germ cell nuclei from staged young adult wild-type animals were scored for the presence of YFP, 3 h after exposure to 100 J/m2 of UV-C or 120 Gy of X-rays. RAD-54 or RPA-1 show a diffuse nuclear staining in control animals, but relocalize into distinct foci following treatment (arrowheads). (b) XPA-1, XPC-1 and MRE-11 are required for RAD-54 foci formation in response to UV-C, but not IR. (d and e) UV-C-induced RPA-1 foci require XPA-1 (d), but not MRE-11 (d) or RAD-54 (e). RAD-54::YFP and RPA-1::YFP foci were quantiﬁed as described in Materials and Methods. Data shown represent the average±S.D. of at least two experiments (nZ15 worms for each experiment)

Cell Death and Differentiation HR is required for UV-C-induced apoptosis in C. elegans L Stergiou et al 901

YFP-tagged RPA-1, the homolog of the largest human RPA Bcl-2 homolog CED-9, nor did it affect the egg laying rate subunit p70, showed foci as early as 30 min after UV-C or IR (Supplementary Figures 5a and b). IR-induced apoptosis still treatment, reaching a plateau 3.5 h later (Figures 2c and d; occurred in xpg-1 mutants, although with reduced levels Supplementary Figures 4a and b). Loss of XPA-1 completely (Figure 3d). As IR-induced apoptosis is normal in other NER blocked the increase of RPA-1 foci following UV, but not IR, mutants (e.g., xpa-1, xpc-1), XPG-1 likely participates in other suggesting that the NER machinery is required for UV-C-, but repair pathways following IR. Indeed, human XPG has been not for IR-induced accumulation of RPA-1 (Figure 2d). suggested to also function in base excision repair, as a In rad-54 mutants, RPA-1::YFP foci numbers were cofactor for hNth1 DNA-glycosylase-AP-lyase during oxida- increased even in the absence of treatment (Figure 2e), likely tive DNA damage repair.24 Remarkably, the increased marking sites of unresolved meiotic recombination intermedi- apoptosis in rad-54(RNAi) animals (Figure 1b) was sup- ates.21 Surprisingly, UV-C and IR treatment did not increase pressed by the loss of xpg-1 function (Figure 3g), suggesting much further the number of RPA-1::YFP foci in rad-54 that XPG-1 acts downstream of, or in parallel to, RAD-54 to mutants (Figure 2e). To distinguish between saturation in promote apoptosis in response to unresolved recombination DNA damage signaling and a requirement for RAD-54 in intermediates. This effect was specific to rad-54,as damage-induced foci formation, we used a spo-11-deficient xpg-1(tm1670) did not suppress the increased apoptosis in background. Both the excess in apoptosis and the high levels rad-51(RNAi) animals (Supplementary Figure 3d). of RPA-1::YFP foci were abrogated in rad-54(ok615);spo- We showed above that XPA-1 is required for UV-C-induced 11(RNAi) animals (Figures 1g and 2e), and exposure to UV-C RAD-54::YFP foci formation. Similarly, foci formation was now led to a robust increase in foci number, suggesting that reduced by twofold or more in xpb-1(RNAi) or xpd-1(RNAi) RPA-1 foci formation occurs upstream of, or in parallel to, animals (Figure 3e). By contrast, loss of XPG-1 function did RAD-54 in the UV response. RPA-1::YFP foci similarly not have any strong effect (Figure 3e). Notably, untreated increased following UV-C in mre-11(ok179) mutants animals had elevated foci numbers in all three cases, likely (Figure 2d). Thus, most RPA-1::YFP foci following UV-C reflecting accumulation of unrepaired lesions. Similarly, loss require the NER machinery and occur upstream of the HR of xpg-1 did not inhibit recruitment of RPA-1::YFP (Figure 2d), components tested here. which we showed to be recruited upstream of RAD-54 upon In summary, our results so far show that components of UV-C (Figure 2e). the HR pathway act downstream of the NER DNA damage Taken together, our findings suggest that recruitment of recognition step to trigger UV-C-induced apoptosis. NER RAD-54 onto sites of UV-C damage does not require the pathway activity leads to focal recruitment of RPA-1 and whole complement of NER genes we analyzed: the DNA- RAD-54 and signaling to the apoptotic machinery. binding protein XPA-1, and the XPB-1 and XPD-1 helicases appear sufficient for this step. The NER components XPG, XPB and XPD are required to promote UV-C-induced apoptosis by recruitment of HR The HUS-1 complex functions downstream of the HR proteins. During NER, initial recognition of the lesion, machinery to promote UV-C-induced apoptosis. We carried out by XPA and XPC, is followed by local previously showed that recruitment of the HUS-1-containing unwinding of the DNA, performed by two DNA helicases 9-1-1 complex in response to UV-C requires XPA-1 with opposite polarities, XPB and XPD.22 This enables the function.11 Similarly, elimination of rad-54 blocked excision step carried out by two structure-specific accumulation of HUS-1::GFP foci following UV treatment endonucleases, XPF and XPG.23 (Figure 3f). By contrast, we observed a modest increase To determine whether NER steps downstream of DNA in foci numbers following IR (Figure 3f). Proteins involved in lesion recognition also play a role in UV-C-induced apoptosis, early stages of HR, therefore, act upstream of and are we analyzed germ cell apoptosis in animals with compromised important for 9-1-1 recruitment in UV response. By contrast, or abolished XPB, XPD or XPG function. As with xpa-1 and lack of xpg-1 did not affect HUS-1::GFP foci formation xpc-1, RNAi knockdown of xpb-1 (Y66D12A.15) and xpd-1 following either UV-C or IR treatment (Figure 3f). Thus, XPG-1 (Y50D7A.2) gave rise to increased basal levels of apoptosis must impinge on the apoptotic signaling pathway at a step (Figures 3a and b). These findings strengthen the notion that downstream of, or in parallel to, 9-1-1 recruitment. in the absence of NER, unrepaired endogenous damage can activate (an)other DNA damage signaling pathway(s) that The HR repair factors are involved in a signaling process lead to apoptosis. xpb-1(RNAi)- and xpd-1(RNAi)-treated to the apoptotic machinery following UV-C. Our finding animals showed reduced apoptosis levels upon both UV-C that HR components are involved in UV-C-induced apoptosis and IR (Figures 3a and b), suggesting that these two helicases led us to speculate on their exact role: are these proteins are also involved in promoting UV-C-induced apoptosis. merely involved in signaling to the apoptotic apparatus, We also analyzed the effect on apoptosis of two likely or do they also participate in the repair of the initial UV-C null alleles of the endonuclease gene xpg-1 (F576B10.6) lesions? (Supplementary Figure 1c). UV-C-induced apoptosis was To address this, we assessed the DNA repair activity in abolished in xpg-1 mutants (Figure 3c). The observed defect mutants defective in either NER or HR. We performed in vivo in xpg-1 (and xpa-1) mutants is not due to a general defect in immunofluorescence to measure the presence of CPDs – one germline physiology, which could reduce the number of cells of the two major types of lesions caused by UV exposure – at that can undergo apoptosis: loss of xpg-1 (or xpa-1) did not defined time intervals in wild-type animals. Pachytene germ reduce apoptosis in animals defective in the antiapoptotic cells showed a maximum CPD signal immediately following

Cell Death and Differentiation HR is required for UV-C-induced apoptosis in C. elegans L Stergiou et al 902

Figure 3 C. elegans NER component XPG-1 is required for UV-C-induced germ cell apoptosis, but not for recruitment of RAD-54. Staged wild-type L1 larvae were raised on bacteria expressing xpb-1 (a), xpd-1 (b) or control dsRNA, and were treated with either 100 J/m2 UV-C or 120 Gy X-rays as young adults. Germ cell corpses were scored at the indicated time points. We could not score the 36-h time point in these animals, as the germ lines started to degenerate. This is possibly owing to the fact that both XPB-1 and XPD-1 are components of TFIIH, and thus might also affect mRNA transcription. (c and d) Apoptosis was scored in xpg-1(tm1670) or xpg-1(tm1682) mutant animals after treatment with 100 J/m2 UV-C (c) or 120 Gy X-rays (d). (e) XPG-1 is not required for UV-C-induced RAD-54 foci formation. RAD-54::YFP foci were quantiﬁed 3 h after treatment with 100 J/m2 UV-C as described in Materials and Methods. (f) RAD-54, but not XPG-1, is required for UV-C-induced HUS-1 foci formation. HUS-1::GFP foci were quantiﬁed in mid–late pachytene germ cell nuclei from staged young adult animals expressing HUS-1::GFP (opIs34) 3 h after exposure to 100 J/m2 of UV-C or 120 Gy of X-rays. Data shown in all cases represent the average±S.D. of three independent experiments (n415 animals for each experiment). (g) Increased levels of apoptosis in rad-54 mutants are suppressed by xpg-1. Germ cell apoptosis was scored 12 and 24 h post the L4/adult molt in staged wild-type (wt), xpg-1(tm1670), rad-54(RNAi) and xpg-1(tm1670);rad-54(RNAi) animals. Data shown represent the average±S.D. of two independent experiments (n420 animals for each experiment)

Cell Death and Differentiation HR is required for UV-C-induced apoptosis in C. elegans L Stergiou et al 903

Figure 4 CPD repair kinetics in NER and HR pathway mutants. (a) Immunofluorescence images of germ cell nuclei from UV-C-irradiated wild-type, rad-54(ok615) and xpa-1(ok698) worms. Gonads were extruded and fixed immediately following UV-C radiation or after 14 h. Images in the upper panel represent the CPD staining (green), the nuclei DAPI staining (magenta) and an overlay of the CPD staining and DAPI in wild-type animals, resulting in a white ring where UV lesions are detected on germ cell chromatin. Although in the absence of NER in xpa-1(ok698) mutants nuclei remain white (lower panel), CPDs are removed in rad-54(ok615) mutants similar to wild type mutants (chromatin turns magenta). (b) CPD signal intensity after irradiation with 100 J/m2 UV-C was determined by fluorescence image analysis of isolated gonads. Intensity before irradiation was defined as 0 and the value immediately after UV-C as 1 for each strain, and later time points were expressed as a fraction of the initial UV-C-induced intensity. At least 10 animals were scored per data point. Error bars represent the S.E.M. Repair kinetics for CPD lesions in transition cell nuclei of wild-type hermaphrodites (t1/2 approx. 10 h) are in good agreement with repair kinetics reported for other systems. xpa-1(ok698) mutants lacking a functional NER show no significant repair, whereas rad-54(ok615) mutants exhibit a response similar to wild type mutants. (c) RAD-54::YFP foci develop slowly and persist for long in UV-C-irradiated wild-type animals. Mid–late pachytene germ cell nuclei from staged young adult wild-type animals were scored for the presence of RAD-54::YFP foci, at 5, 10, 20 min, 1, 2, 4, 8 and 16 h post 100 J/m2 of UV-C treatment

UV-C (Figures 4a and b), which gradually disappeared with a not pro-apoptotic. However, their processing by NER can half-life of approximately 10 h. These repair kinetics fall within lead, in a fraction of cases, to the generation of other types of the range reported for other systems.25,26 Interestingly, CPDs DNA damage (e.g., DSBs, or long stretches of ssDNA). These were cleared at the same rate in rad-54(ok615) mutants as in are in turn recognized and processed by HR, leading to repair wild type (Figure 4b), consistent with the notion that RAD-54 is and, if necessary, apoptosis of the damaged cell (Figure 5). not involved in CPD lesion repair. By contrast, xpa-1 mutants showed a greatly reduced ability to remove CPDs (Figure 4b), Discussion conﬁrming the role of C. elegans xpa-1 in NER. Unlike the relatively rapid appearance and repair of CPDs, In this paper, we took advantage of the powerful genetics in RAD-54::YFP foci developed more slowly, peaking 2 h after C. elegans to investigate in detail the signaling pathways that treatment, and also persisted for much longer (Figure 4c). activate apoptosis in response to UV-C light. We show that Their perdurance might explain the apparent conundrum that apoptosis requires the coordinated action of two repair UV-C-induced apoptosis persists for much longer than the systems, the NER and HR pathways, and present a molecular CPDs (over 36 versus 10–20 h): it is not the CPDs model that explains this novel ﬁnding. themselves, but rather some type of DNA intermediates, How does UV-C cause apoptosis in C. elegans? A simple recognized or marked by RAD-54, which promote apoptosis. explanation would be that UV-C directly causes double-strand How might these two types of damage be linked? We breaks, which could activate the well-characterized ATM suggest the following model: UV lesions by themselves are (/ATR)/CHK2/p53 pathway previously shown to respond to

Cell Death and Differentiation HR is required for UV-C-induced apoptosis in C. elegans L Stergiou et al 904

Figure 5 UV-C-induced apoptosis requires the action of both the NER and HR pathways. (a) Summary of the genetic requirements for the recruitment of NER and HR pathway components onto DNA lesions, as well as the induction of apoptosis following UV-C treatment. (b) Molecular model for UV-C-induced apoptosis in C. elegans. The NER machinery (blue) transforms a fraction of the UV-C lesions into other types of DNA damage (e.g., double-strand breaks). These, in turn, are then recognized by the HR machinery (green), leading to DNA repair and/or apoptosis of the damaged cell. Remarkably, XPG-1 deﬁnes a new branch that acts downstream of 9-1-1 to induce apoptosis

such lesions.27,28 However, while mutagenic and cytotoxic, suggests that the damage recognized by the HR repair UV-C lesions are not pro-apoptotic per se: mutants in the DNA system is a product of NER activity. damage recognition components of the NER machinery, On the basis of these results, an attractive model for xpa-1 and xpc-1, are defective in UV-C-induced apoptosis.11 UV-C-induced apoptosis would be that the NER machinery The requirement for these components suggests that their transforms a fraction of the UV-C lesions – wittingly or substrates, CPDs and 6-4PPs, are the relevant lesions, and unwittingly – into DSBs. These, in turn, are recognized by the not DSBs. Furthermore, induction of apoptosis requires both HR pathway and lead to apoptosis via the pathway that we recognition and processing of these lesions by the NER and others described previously (Figure 5).11,30 Consistent machinery, as the DNA helicases XPB-1 and XPD-1 and the with this model, the lesions or DNA structures positive for the ssDNA endonuclease XPG-1 were also important for the presence of RAD-54 only appear progressively, over a few activation of UV-C-induced apoptosis (Figure 3). A similar hours, in response to NER activity. How the latter can requirement for XPB and XPD has also been reported in generate DSBs remains elusive. Might they happen acciden- human primary ﬁbroblasts, as part of a p53-mediated tally when the NER machinery excises a stretch of the apoptotic pathway.29 damaged strand? Assuming two CPDs per 104 bases at The requirement of a functional NER pathway for UV-C- 100 J/m2 (compare Boyd et al.31), one haploid worm genome induced apoptosis raises the question as to whether it is the (108 bp) would initially harbor 4 Â 104 lesions. Theoretically, repair process per se, or rather its output (the resulting DNA this leaves a risk of almost 500 occurrences per cell of structures) that triggers apoptosis. Interestingly, the NER two lesions on opposing DNA strands within 30 nt, which, pathway is not the only repair pathway involved. We showed when processed simultaneously, could result in a DSB. here that mutations in the HR pathway genes mre-11 and For comparison, Vilenchik et al.32,33 estimated that in one rad-54 also abrogate UV-C-induced cell death (Figures 1a and f). mammalian cell cycle, 1% of single-strand lesions are This lack of apoptosis in a post-mitotic cell population converted into 50 endogenous DSBs. (mid–late pachytene germ cells), in which UV-C lesions Although attractive, this model cannot fully explain a cannot be converted into DSBs via replication, strongly number of intriguing observations. First, we previously

Cell Death and Differentiation HR is required for UV-C-induced apoptosis in C. elegans L Stergiou et al 905 showed that UV-C and IR activate different sets of checkpoint CPD staining and quantification. A modified fixation protocol was applied kinases.11 However, we propose here that both treatments to preserve/create denatured DNA structures for CPD staining, as this antibody specifically binds to CPDs in ssDNA.40 Adult hermaphrodites were irradiated with activate apoptosis via DSBs. Perhaps, the nature of the DSBs 2 or of the associated protein complexes is different in the two 100 J/m UVC and, at the indicated time points, were dissected in M9 (0.5 mM levamisole) to extrude the gonads and fixed in M9/3% PFA for 30 min. Fixed worms cases. Second, we showed that while XPG-1 is required for were transferred to poly-lysine-coated slides, freeze-cracked and permeabilized in UV-C-induced apoptosis, it is not needed for the generation of PBS/Tween-20 0.1% for 3 Â 10 min. For DNA denaturation, slides were incubated RAD-54-positive foci; furthermore, loss of xpg-1 function can in PBS/0.07 N NaOH for 8 min and washed in PBS/Tween-20 0.1% for 3 Â 10 min suppress the increased apoptosis in non-irradiated worms before blocking (1 h at RT) and incubation with 1 : 500 anti-CPD monoclonal defective for rad-54. At which level precisely XPG-1 functions antibody (MBL International, Woburn, MA, USA; product no. D194-1) for at least 6 h 1 to promote apoptosis is itself an intriguing topic. at 4 C. Samples were washed and incubated (1 h at RT) with Alexa Fluor 488 goat 34,35 anti-rabbit (Invitrogen) as secondary antibody (1 : 500) and counterstained with Muzi-Falconi and co-workers have suggested a func- DAPI (200 ng/ml) before mounting in Prolong Gold (Invitrogen). Blocking and tional link between NER and checkpoint activation in both antibody incubations were in antibody buffer/10% goat serum. yeast and mammalian cells, in which processing of UV lesions Fluorescent images were captured as described above with defined exposure by the XPA-1 homolog enables 9-1-1 complex recruitment settings. For quantification of CPD signal intensity, a macro was created and run in and activation of the ATR kinase.34 Toussaint et al.36 have ImageJ (version 1.42q; http://rsb.info.nih.gov/nih-image/), allowing for automated characterized the co-participation of HR components in measurements after definition of a region of interest (ROI) in the immunofluorescence pictures. Briefly, an ROI was drawn around approximately 100 germ cell rendering G2-phase yeast cells more resistant to UV nuclei of the transition/early meiotic region. A mask was defined from a binary irradiation. Recent evidence might shift the concept of threshold image of the DAPI staining to account for DNA containing areas and individual repair pathways toward that of a DNA damage applied to the anti-CPD image. Average anti-CPD signal intensity over DAPI- response network, in which DNA repair factors interact to positive areas was compared with average intensity of the background within the drive a coordinated response toward genome integrity and cell ROI, and expressed as difference. survival,37 or towards cell death. Our findings suggest the existence of a cross-talk between Foci quantification. Staged young adult hermaphrodites were treated with either 100 J/m2 of UV-C light or 120 Gy of X-rays, and gonads were dissected at the NER and HR repair pathways, and assign a role to several indicated time points. RAD-54::YFP and RPA-1::YFP foci formation was quantified DNA repair components in apoptotic signaling. As repair by counting the number of bright foci present in mid–late pachytene germ cells, in pathways are highly conserved through evolution, the inter- one focal plane within 100 mm from the first oocyte. Fluorescent images were action between the NER and the HR pathways that we captured with a Leica DMRA2 microscope equipped with an ORCA-ER digital CCD describe here for C. elegans might also occur in vertebrates, camera and were processed with an Openlab software. including humans. RNAi constructs. Fragments corresponding to exonic sequences of xpb-1 (Y66D12A.15) and xpd-1 (Y50D7A.2) were amplified by PCR from a wild-type cDNA Materials and Methods library using primers listed in Supplementary Table S2, and cloned into the L4440 Genetics. All strains were grown at 201C on NGM agar seeded with Escherichia RNAi vector. The constructs were used to transform HT115(DH3) bacteria, which coli OP50. The Bristol N2 strain was used as the wild-type strain. Mutant alleles were subsequently used to feed worms in the RNAi experiments. For the rest of the used are listed below: LGI: atm-1(gk186),11 hus-1(op244),38 xpg-1(tm1670) (this RNAi clones in this study, we used the Ahringer RNAi library. A standard gfp study), xpg-1(tm1682) (this study), xpa-1(ok698),11 rad-54(ok615) (this study), sequence cloned into L4440 vector or the empty vector was used as controls for the rad-54(tm1268) (this study); LGIV: xpc-1(ok734),11 spo-11(ok79);39 and LGV: RNAi experiments. atl-1(tm853),30 mre-11(ok179),12 chk-2(gk212).11 Transgenes: opIs257 (RAD- 54::YFP) and opIs263 (RPA-1::YFP) (this study). Essential mutations were Transgenic worms. Genomic fragments corresponding to rpa-1 and rad-54 maintained as balanced strains: rad-54(ok615)/hT2[qIs51](IV;V), rad-54(tm1268)/ promoter and ORF, as well as the 30-UTR region were separately amplified by PCR hT2[qIs51](IV;V), spo-11(ok79)/nT1[n754 let](IV;V), atl-1(tm853)/nT1[qIs51](IV;V) from N2 genomic DNA using primers (Supplementary Table S2) that added the and mre-11(ok179)/nT1[n754 let](IV;V). Additional information on mutations cited appropriate restriction sites. The amplified fragments were cloned into the pLN022 here can be obtained at www.wormbase.org. expression vector upstream of yfp to generate pLS69 (RPA-1::YFP) or pLS57 (RAD-54::YFP). Low-copy transgenic worms were then generated by ballistic transformation. Sequences of all used plasmids are available upon request. Germline apoptosis. Staged young adult worms (12 h post the L4/adult molt) were exposed to different doses of UV-C light (254 nm) (J/m2) or X-rays (Gy). A Stratalinker UV crosslinker, model 1800 (Stratagene, Basel, Switzerland) and an Conflict of interest Isovolt 160/225/320/450 HS X-ray machine (Rich. Seifert & Co., Ahrensburg, Germany) were used to deliver the appropriate doses. Corpses were scored in the The authors declare no conflict of interest. meiotic region of one gonad arm at indicated time points using Nomarski optics. For the RNAi experiments, staged L1 larvae were transferred onto plates containing 2 mM IPTG seeded with E. coli strain HT115(DH3) expressing the respective RNAi Acknowledgements. We thank Professor H Naegeli and Dr A Gartner for clone, and scored as young adults for germline apoptosis. critical comments on our manuscript, and members of the Hengartner lab for discussions. This work was supported by the Kanton of Zurich, the Swiss National Science Foundation, the Ernst Hadorn Foundation and the Josef Steiner Cancer Immunocytochemistry. For antibody staining of gonads, young adult Research Foundation. RE was supported by an MD-PhD fellowship from the Swiss hermaphrodites were dissected and fixed in 3% PFA/0.1 M K2HPO4 (pH 7.2) for National Science Foundation. We also thank Dr A Gartner for providing us with the 50 min at room temperature, followed by a 10-min incubation in 100% methanol on RAD-51 antibody, and the Caenorhabditis Genetics Center, which is funded by the ice. Gonads were blocked in 5% BSA/PBS–Tween-20 0.1% for 1 h, followed by National Institute of Health (NIH), National Center for Research Resources (NCRR) 15 incubation with 1 : 50 anti-RAD-51 polyclonal antibody overnight at 41C. Alexa as well as Shohei Mitani of the National Bioresource Project in Japan for strains. Fluor 594 goat anti-rabbit IgG (Invitrogen, Basel, Switzerland) was used as secondary antibody (1 : 500). The tissues were co-stained with DAPI before mounting. Fluorescent images were captured with a Leica DMRA2 microscope 1. Hanahan D, Weinberg RA. The hallmarks of cancer. 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Cell Death and Differentiation